In focus: Triple Negative Breast Cancer

‘Triple negative breast cancer’ or TNBC, as the name suggests, is a classification of breast cancers lacking the expression of estrogen receptor (ER) and progesterone receptor expression as well as amplification of the human epidermal growth factor receptor 2 (HER2).

Unlike other breast cancer types, treating TNBC is a challenge mainly because of the absence of well-defined molecular targets and because of disease heterogeneity. Currently, neoadjuvant chemotherapies are in use to treat TNBC patients. Some, around 30%, patients respond completely to neoadjuvant chemotherapy and have good outcomes after surgery. However, if there is a residual disease after therapy, outcomes are poor.

Therefore, current focus of the field is to first understand the complexity of the disease, both at genomic and molecular level and look for targets. Also, several combination chemotherapies are currently under trial to determine the efficacy, overall response rate, progression-free survival and other relevant factors for patients suffering with different forms of TNBC.

Recently, in the San Antonio Breast Cancer Symposium (SABCS 2012), several abstarcts related to TNBC research, both clinical and pre-clinical. Here is a compilation of some of the abstracts and their relevance in the field of TNBC research:

In order to better understand the complexity of TNBC, an integrative and comprehensive genomic and molecular analysis is required. The analysis would give important cues to developing and administering effective therapeutic agents. The group has compiled an extensive number of TNBC gene expression profiles and initiated molecular subtyping of the disease. Differential GE was used to designate 25 TNBC cell line models representative of the following subtypes:

two mesenchymal subtypes with high expression of genes involved in differentiation and growth factor pathways (M and MSL);

an immunomodulatory (IM) type;

a luminal subtype driven by androgen signaling (LAR)

The pharmacological drugs were chosen on the basis of the genetic pathways active in the cell lines with the abovementioned TNBC subtypes. It was observed that BL1 and BL2 subtype cell lines respond to cisplatin. Mesenchymal, basal, and luminal subtype lines with aberrations in PI3K signaling and have the greatest sensitivity to PI3K inhibitors.

The LAR subtype cell lines express AR and are uniquely sensitive to bicalutamide (AR antagonist). The experiment was a proof-of-concept that the best therapy could be based on TNBC subtypes.

The group has also developed a web-based subtyping tool referred to as “TNBCtype,” for candidate TNBC tumor samples using our gene expression metadata and classification methods. The approach would enable alignment of TNBC patients to appropriate targeted therapies.

The abstract is on the same lines, TNBC heterogeneity that is. The concept of clonal heterogeneity in cancers, the spatial and temporal variation in clonal composition, is the focal point of the discussion. The group has developed next generation sequencing approaches and applied them to the understanding of mutational and clonal composition of primary TNBC. They have demonstrated that both mutational composition and clonal structure of primary TNBC is in fact a complete spectrum, a notion that is far from the previous one that stated TNBC to be a distinct disease. The authors add “clonal analysis suggests a means by which the genetic complexity might be reduced by following patient evolution over time and space.” The specific implications of the mutational and transcriptome landscapes of TNBC in relation to possible disease biologies were discussed in the symposium.

In the absence of hormone receptors and hence lack of targets, Neoadjuvant chemotherapy (NAC) is increasingly used in patients with TNBC. NAC can induce a pathologic complete response (pCR) in ∼30% of patients which portends a favorable prognosis. In contrast, patients with residual disease (RD) in the breast at surgical resection exhibit worse outcomes. The authors hypothesize that “profiling residual TNBC after NAC would identify molecularly targetable lesions in the chemotherapy resistant component of the tumor and that the persistent tumor cells would mirror micro-metastases which ultimately recur in such patients.” The researchers utilized targeted next generation sequencing (NGS) for 182 oncogenes and tumor suppressors in a CLIA certified lab (Foundation Medicine, Cambridge, MA) and gene expression profiling (NanoString) of the RD after NAC in 102 patients with TNBC. The RD was stained for Ki67, which has been reported to predict outcome after NAC in unselected breast cancers. Out of the 89 evaluable post-NAC tumors, 57 (64%) were basal-like; 19% HER2-enriched; 6% luminal A; 6% luminal B and 5% normal-like. Of 81 tumors evaluated by NGS, 89% demonstrated mutations in TP53, 27% were MCL1-amplified, and 21% were MYC-amplified.

NGS identified 7 patients with ERBB2 gene amplification. NGS could assist in the identification of ERBB2-overexpressing tumors misclassified at the time of diagnosis.

Amplifications of MYC were independently associated with poor recurrence-free survival (RFS) and overall survival (OS). In contrast to the earlier notion, high post-NAC Ki67 score did not predict poor RFS or OS in this predominantly TNBC cohort.

The authors concluded that “the diversity of lesions in residual TNBCs after NAC underscores the need for powerful and broad molecular approaches to identify actionable molecular alterations and, in turn, better inform personalized therapy of this aggressive disease.”

Reiterating the greatest challenge of the TNBC treatment, no targeted agents currently exist against TNBC. The group at UCSF has discovered that TNBC frequently express high levels of the MYC proto-oncogene. The discovery has led them to identify new “synthetic-lethal” strategies to selectively kill TNBC with MYC overexpression. “Synthetic lethality arises when a combination of mutations in two or more genes leads to cell death, whereas a mutation in only one of these genes has little effect. Using this strategy, we can take advantage of the elevated MYC signaling in TNBC to selectively kill them, while sparing normal tissues in which MYC is expressed at much lower levels”

The researchers performed a shRNA synthetic-lethal screen in the human mammary epithelial cells (HMEC), to identify new molecules, such as cell cycle kinases, which when inhibited can preferentially kill TNBC cells. A high-throughput screen of ∼2000 shRNAs, that target the human kinome (∼ 600 kinases) when performed, led to the identification of 13 kinases whose inhibition by >1 shRNAs gave rise to >50% inhibition of cell growth. ARK5 and GSK3A were the two other kinases that were shown to have a synthetic-lethal interaction with MYC. Since these two kinases have been identified in other studies, it gives validity to the ability to the methods of Goga etal in identifying synthetic-lethal targets. The group is currently characterizing and validating the 11 novel targets identified in this screen, using human cancer cell lines as well as mouse cancer models to determine the impact of inhibiting these targets on triple-negative breast cancer development and proliferation.

Case 3: pl. Cite for myc, Lev-Ari, Part 1,2,3,4 – here part one only, and Larry’s Directing for Genomics

I commissioned Larry to write following Part 1,2,3,4, the mortar for the bricks i laid out in 1,2,3,4 — his emphasis on glucose and Cancer is very important. Another aspect would be vascularization and cancer, may be YOU wish to have a post on that with imaging examples on vascularization.